Malaria is caused by parasites of the genus Plasmodium. According to the Centers for Disease Control, malaria ranks second in Africa as the greatest cause of death from infectious diseases, after HIV/AIDS. It ranks fifth worldwide, after respiratory infections, HIV/AIDS, diarrheal diseases, and tuberculosis. Plasmodium falciparum, one of at least eleven known Plasmodium parasites that attack humans, causes a particularly severe infection characterized by sequestration of the parasite in vital organs and deep tissues where it can evade the immune system.
There is no effective malaria vaccine available. Recent strategies target the Plasmodium falciparum circumsporozoite protein (CSP), which is critical for the pathogenesis of the parasite. Currently, a vaccine called RTS,S (GlaxoSmithKline), composed of a portion of CSP, is in Phase III clinical trials. CSP is a protein monomer that can be broadly described as having three regions—the N-terminal region, the central repeat region, and the C-terminal region. The N and C-terminal regions contain crucial protective regions important for parasite invasion, and the central region contains highly conserved immunodominant tetrapeptide repeats. The vaccine RTS,S does not include the N-terminal region of CSP. It is composed of a portion of the CSP central repeat and the C-terminal region, linked to hepatitis B surface antigen. Recent reports indicating that the N-terminal region of CSP is immunogenic suggest that a vaccine strategy utilizing a CSP molecule having the N-terminal region would be superior.
Development of a manufacturing scale purification process to make recombinant CSP in amounts that meet the needs for vaccine research and production presents challenges. The N-terminal region of CSP is highly susceptible to degradation. Furthermore, CSP dimerizes due to the formation of covalent intermolecular disulfide bonds that involve a free cysteine near the monomer's N-terminus. CSP also forms higher molecular weight aggregates. Present purification schemes provide recombinant CSP monomer lacking the N-terminal region, or they generate low yields of intact CSP. Denaturation to eliminate dimers and aggregates has required refolding, which is complicated by the presence of two disulfide bonds involving four cysteine residues in the C-terminal region of CSP. These disulfide bonds are critical for the structure and function of the C-terminal protective region; disrupting them has been shown to destroy CSP's ability to bind to liver cells. Furthermore, additional denaturing and refolding steps are burdensome, costly, reduce yield, and are challenging to scale up for use with large fermentation batches. Therefore, scalable purification methods for obtaining high quality recombinant CSP at high yields are needed.